Specific antibody directed to active hepatocyte growth factor activator and method for using the same

ABSTRACT

An antibody which recognizes an active hepatocyte growth factor activator (HGFA) and does not substantially recognize inactive HGFA is provided. Also disclosed is a monoclonal antibody thereof, and a hybridoma cell line for producing the monoclonal antibody. There is further provided a method for measuring active HGFA using the antibody, and a method for detecting a disease, by detecting or measuring active HGFA using the antibody.

This application is a divisional of application Ser. No. 10/000,096,filed Dec. 4, 2001, currently pending.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an antibody or a monoclonal antibodyused for specifically measuring active hepatocyte growth factoractivator (HGFA), a method for using the same and a measurement kit. Thepresent invention also relates to a method for detecting a disease ofpatient in a pathological condition, in particular, organ inflammation,glomerular nephritis, cancer, myocardial infarction, angina pectoris orthrombosis, by using the active HGFA as an index and further relates toa method for collecting a biological component and blood, which issuitable for performing the method.

2. Description of the Related Art

Protease (protein decomposition enzyme) is a protein having a functionof hydrolyzing a peptide bond in a protein or a peptide and it is deeplyinvolved in organic functions and, at the same time, plays an importantrole in maintenance of homeostasis thereof. For example, variousproteases existing in blood themselves constitute dense cascades andcontrol coagulation and fibrinolysis of blood.

A hepatocyte growth factor activator (hereinafter abbreviated as “HGFA”)is known as a kind of serine protease having a serine residue in itsactive center (Miyazawa et al., J. Biol. Chem., 268, pp. 10024-10028,1993). Unlike common proteases in blood involved in the bloodcoagulation/fibrinolysis system cascades, HGFA has a uniquecharacteristic of acting on a hepatocyte growth factor (hereinafterabbreviated as “HGF”), which is known as a cytokine involved inhepatocyte growth or organ regeneration (Naka et al., J. Biol. Chem.,267, pp. 20114-20119, 1992), to specifically and limitedly decompose itand thereby activate it (Shimomura et al., Cytotechnology, 8, pp.219-229 (1992)). However, as for the action of HGFA, only the activationof HGF is known in animal model experiments using rats or in vitroexperiments (Miyazawa et al., J. Biol. Chem., 271, pp. 3615-3618, 1996),and its roles and functions in human pathologic conditions and therelationship of its blood level and pathologic conditions have not beenknown at all.

As HGFA, there are known one showing a molecular weight of about96,000-98,000 determined by SDS-PAGE (hereafter, abbreviated as “98 kDaHGFA”) and one showing a molecular weight of about 34,000-38,000(hereafter, abbreviated as “36 kDa HGFA”), which is a peptide on the Cterminus side of the protein provided by limited proteolysis at a bondbetween arginine at a position of 372 and the valine at a position of373, which are counted from the translation initiation amino acid. Thevariation in the molecular weights is caused by heterogeneity in thebonding amount of sugar chains and differences in measured molecularweight values attributable to whether the measurement by the SDS-PAGEmethod was performed under reducing or non-reducing condition, and theseare essentially the same protein. Further, each HGFA usually exists asan inactive substance in blood, but is activated by limited proteolysisat a bond between the arginine at position of 407 and isoleucine at aposition of 408, which are counted from the translation initiation aminoacid, and thus single chain HGFA is converted to double chain HGFA,which is heterodimerized with a disulfide bond. This activated HGFA isconsidered to specifically activate the substrate, HGF (Shimomura etal., J. Biol. Chem., 268, pp. 22927-22932, 1993).

To analyze the relationship between the HGFA blood level andpathological conditions, active HGFA existing in biological componentsuch as human tissues, humors or blood needs to be specificallymeasured. Further, to specifically detect or measure active HGFA, it isessential to obtain an antibody, particularly preferably a monoclonalantibody, that recognizes active HGFA extremely specifically, but doesnot substantially recognize inactive HGFA. However, any antibody havingsuch a characteristic has not known at all so far. Further, there is nomethod or kit specifically used for measuring active HGFA existing in abiological component such as human blood.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide anantibody that specifically recognizes active HGFA and does notsubstantially recognize inactive HGFA, a method for measuring activeHGFA by using the antibody and a method and kit for detecting a diseaseassociated with active HGFA.

The inventors of the present invention constructed a system forquantifying HGFA in human blood to analyze the relationship between theHGFA blood level and various human diseases as for human pathologicalconditions. First, they constructed an enzyme-labeled antibodymeasurement system using the double antibody sandwich method(enzyme-linked immunosorbent assay, abbreviated as “ELISA measurementsystem” hereafter) by using 7E10, P1-4, A-1, A-6, A-23, A-32, A-51,A-75, which are known as existing mouse monoclonal antibodies directedto HGFA (Miyazawa et al., J. Biol. Chem., 271, pp. 3615-3618, 1996).Further, they analyzed reactivity of HGFA monoclonal antibodies in blood(plasma or serum) of healthy subjects and patients with various diseasesincluding organ diseases. However, uniformly strong reactivity wasobserved in all human blood including that of normal subjects by theELISA measurement system using these monoclonal antibodies, and noreactivity specific to human diseases, that is, no marked changes in theHGFA blood level could be detected. Therefore, the inventors of thepresent invention analyzed reactivity to HGFA of the existing monoclonalantibodies directed to HGFA to investigate causes therefor.

First, since there are two forms of HGFA, active and inactive types,reactivity to HGFA of monoclonal antibodies directed to respective HGFAwas analyzed. More specifically, active HGFA and inactive HGFA wereadhered to a solid phase plate, and reactivity between the HGFA adheredto the solid phase and respective monoclonal antibodies was analyzed. Asa result, it was found that these monoclonal antibodies had acharacteristic of being reactive to both of active HGFA and inactiveHGFA. These results suggested that, in the ELISA method using existingmonoclonal antibodies, inactive HGFA, which is considered to constantlyexist in blood in a large amount (Shimomura et al., J. Biol. Chem., 268,pp. 22927-22932, 1993), is measured, or both of a trace amount of activeHGFA and inactive HGFA are measured. Thus, it was found that the ELISAmeasurement system using existing monoclonal antibodies could not revealthe relationship between HGFA and human diseases at all.

Then, paying attention to the fact that HGFA exists in two forms, activeand inactive types, the inventors of the present invention specificallydetected an activated state of HGFA existing in a human living bodiesand examined the relationship between various human diseases and theamount of active HGFA in blood of patients with those diseases. As aresult of their assiduous studies, they successfully produced polyclonalantibodies and monoclonal antibodies that recognize active HGFA but donot substantially recognize inactive HGFA for the first time. Further,they accomplished a method and kit that react with active HGFA existingin a human biological component but do not substantially react withinactive HGFA, that is, a method and kit for specifically measuringactive HGFA, by utilizing an immunoassay using the antibodies.

Further, the inventors found for the first time that, when the methodand kit for specifically measuring active HGFA of the present inventionare used, there could be observed a markedly increased amount of activeHGFA in blood of patients with organ derangement including glomerularnephritis and cancer patients compared with that of healthy subjects.Further, they also found for the first time that thrombosis such asangina pectoris, myocardial infarction and cerebral infarction could beaccurately predicted by using the increase in the amount of active HGFAin blood as an index.

Meanwhile, when the amount of active HGFA existing in a biologicalcomponent including human blood is measured, there is required a methodof stably collecting active HGFA existing therein with goodreproducibility. The inventors of the present invention examinedaddition of various protease inhibitors and so forth in this method andfound that active HGFA could be extremely stably measured with goodreproducibility by adding argatroban, which is a selective thrombininhibitor. Further, as a result of many detailed investigations, theyalso found that argatroban was effective even when human blood such aswhole blood, serum or plasma was collected and that an extremelyfavorable result could be obtained by using citrated plasma among bloodcomponents.

The present invention was accomplished based on the above findings andprovides the followings.

-   -   (1) An antibody that recognizes an active hepatocyte growth        factor activator (HGFA) and does not substantially recognize        inactive HGFA.    -   (2) The antibody according to (1), which shows a dissociation        constant of 1×10⁻⁸ M or lower for active HFGA.    -   (3) The antibody according to (1) or (2), which is a monoclonal        antibody.    -   (4) The antibody according to (3), which recognizes active HGFA        showing a molecular weight of about 34,000-98,000 determined by        the SDS-PAGE method and does not substantially recognize        inactive HGFA.    -   (5) The antibody according to (4), which recognizes active HGFA        showing a molecular weight of about 34,000-38,000 determined by        the SDS-PAGE method.    -   (6) The monoclonal antibody according to (4), which is produced        by a hybridoma of an accession number FERM BP-7779.    -   (7) A monoclonal antibody that recognizes active HGFA activated        by limited proteolysis of inactive HGFA, which is a precursor of        active HGFA, between arginine at a position of 407 and        isoleucine at a position of 408 counted from a translation        initiation amino acid of inactive HGFA, and does not        substantially recognize inactive HGFA    -   (8) A monoclonal antibody that recognizes active HGFA and does        not substantially recognize inactive HGFA and a complex of        active HGFA and a protease inhibitor.    -   (9) A hybridoma cell line that produces a monoclonal antibody        according to any one of (3) to (8).    -   (10) A method for measuring active HGFA, comprising the step of        measuring the active HGFA specifically by an immunological        method using one or more kinds of antibodies according to any        one of (1) to (8).    -   (11) The method according to (10), wherein a specimen to be        measured for active HGFA is a biological component collected        from a subject or test animal suspected of having a disease.    -   (12) The method according to (11), wherein the disease is organ        inflammation, glomerular nephritis, cancer, myocardial        infarction, angina pectoris, cerebral infarction or thrombosis.    -   (13) A method for detecting a disease, comprising the step of        detecting or measuring active HGFA in a biological component        collected from a subject suspected of having a disease.    -   (14) The method according to (13), wherein the disease is        selected from the group consisting of organ inflammation,        glomerular nephritis, cancer, myocardial infarction, angina        pectoris, cerebral infarction and thrombosis.    -   (15) The method according to (13) or (14), wherein the        biological component is blood or a fraction or processed product        thereof.    -   (16) The method according to (15), wherein the biological        component is plasma.    -   (17) The method according to (16), wherein the plasma is        citrated plasma.    -   (18) The method according to any one of (13) to (17), wherein        argatroban is added to the biological component.    -   (19) A kit for detecting or measuring active HGFA, which        comprises one or more kinds of antibodies according to any one        of (1) to (8).    -   (20) The kit according to (19), which is used for diagnosis of        disease selected from the group consisting of organ        inflammation, glomerular nephritis, cancer, myocardial        infarction, angina pectoris, cerebral infarction and thrombosis.    -   (21) The kit according to (19) or (20), which is used to measure        active HGFA in a biological component collected from a subject        suspected of having a disease.    -   (22) The kit according to any one of (19) to (21), wherein        active HFGA is detected or measured by immunostaining.    -   (23) A blood collection tube for collecting serum, plasma or        whole blood, which is added with argatroban.    -   (24) The blood collection tube according to (23), which is used        to collect serum, plasma or whole blood to be used for        measurement of active HGFA.

In the present specification, a monoclonal antibody that recognizesactive HGFA and does not substantially recognize inactive HGFA may bereferred to as “active HGFA specific monoclonal antibody”, and apolyclonal antibody that recognizes active HGFA and does notsubstantially recognize inactive HGFA may be referred to as “active HGFAspecific polyclonal antibody”. Further, these antibodies may becollectively referred to as “active HGFA specific antibody”. Further,“recognizing active HGFA” means bonding to active HGFA through anantigen/antibody reaction and preferably means that a dissociationconstant for active HGFA is 1×10⁻⁸ M or lower, more preferably 1×10⁻⁹ Mor lower.

“Not substantially recognizing inactive HGFA” means not substantiallybonding to inactive HGFA through an antigen/antibody reaction. Morespecifically, “not substantially recognizing inactive HGFA” means thatinactive HGFA cannot be detected by a usual immunoassay and that adissociation constant for inactive HGFA is 1×10⁻⁵ M or higher.

According to the present invention, there are provided monoclonal andpolyclonal antibodies that specifically bond to active HGFA and do notbond to inactive HGFA. The antibodies of the present invention can beused for specific measurement and detection of active HGFA.

The method for specifically measuring active HGFA of the presentinvention can be used for diagnosis of various diseases reflected inblood level of active HGFA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows reactivities of monoclonal antibody to active HGFA andinactive HGFA.

FIG. 2 shows reactivity between a complex of active HGFA and proteaseinhibitor and a monoclonal antibody.

FIG. 3 shows reactivities between active HGFA and inactive HGFA andmonoclonal antibody measured by an active HGFA measurement system. FIG.3A shows the concentration of HGFA in the range of 0-500 ng/ml. In FIG.3B, the HGFA concentration range of 0-55.6 ng/ml shown in FIG. 3A isenlarged.

FIG. 4 shows a histogram of amounts of active HGFA in sera of healthysubjects.

FIG. 5 shows storage stability of active HGFA value in serum, citratedplasma, heparin plasma and EDTA plasma, and effect of addition ofargatroban.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be explained in detail hereafter.

<1> Immunogen and Screening Antigen for Production of Specific AntibodyDirected to Active HGFA

The term “active HGFA” used in the present invention refers to asubstance having an ability to act on its substrate, hepatocyte growthfactor (HGF), to convert its inactive type, single chain HGF (Naka etal., J. Biol. Chem., 267:20114-20119 (1992)), into its active type,double chain HGF. The single chain HGF refers to a protein obtained froma precursor protein translated from the HGF gene by removal of thesignal peptide, and the double chain HGF refers to a heterodimerconsisting of two chains formed by limited proteolysis of HGF betweenarginine at a position of 494 and valine at a position of 495 countedfrom the translation initiation amino acid and heterodimerized with adisulfide bond. The activity of HGFA refers to an activity of convertinginactive single chain HGF into active double chain HGF.

Active HGFA includes, specifically, HGFA obtained by activation of theHGFA precursor protein disclosed in Japanese Patent Laid-openPublication (Kokai) No. 6-153946 through limited proteolysis betweenarginine at a position of 407 and isoleucine at a position of 408, whichare counted from the translation initiation amino acid. This active HGFAnormally shows a molecular weight of about 34,000-98,000 determined bythe SDS-PAGE method, and there are known active HGFAs obtained bylimited proteolysis of the N-terminus portion and showing variousmolecular weights (Mitsubishi Kasei R & D Review, 8 (1), 26-35 (1994)).As examples of such HGFAs, there are one showing a molecular weight ofabout 98,000 as determined by SDS-PAGE under a reducing condition, onecorresponding to a C-terminus side peptide of the same obtained bylimited proteolysis between arginine at a position of 88 and the alanineat a position of 89, which are counted from the translation initiationamino acid, and showing a molecular weight of about 80,000, and onecorresponding to a C-terminus side peptide obtained by limitedproteolysis of the same between the arginine at a position of 372 andvaline at a position of 373, which are counted from the translationinitiation amino acid, and showing a molecular weight of about 36,000.The aforementioned one showing a molecular weight of about 98,000 mayshow a molecular weight in the range of about 96,000 to 98,000, and theaforementioned one showing a molecular weight of about 36,000 may show amolecular weight in the range of about 34,000 to 38,000, depending onheterogeneity of the amount of bonded sugar chains, method of molecularweight measurement or purification method. Therefore, active HGFAreferred to herein is not limited by its molecular weight, and includesthose having the ability to activate its substrate, HGF.

As active HGFA or inactive HGFA used as immunogen, used for screening ofmonoclonal antibody and so forth, there may be used those purified fromblood of human according to the method of Shimomura et al. (J. Biol.Chem., 268:22927-22932 (1993)) and so forth. Further, it is alsopossible to use HGFA that is a recombinant protein obtained by usingHGFA cDNA disclosed in Japanese Patent Laid-open Publication Nos.6-153966 and 6-153946, U.S. Pat. No. 5,466,593 and U.S. Pat. No.5,677,164 and a microorganism such as Escherichia coli, an insect cell,yeast, an animal cell or an animal.

In order to obtain an active HGFA specific antibody, in particular, itis required to prepare active HGFA of high purity not containinginactive HGFA and inactive HGFA of high purity not containing activeHGFA as an antigen for immunization or an antigen for screening. Forthese purposes, recombinant proteins obtained by using HGFA cDNA arepreferred as active HGFA and inactive HGFA used in the presentinvention. For example, recombinant protein HGFA can be obtained byinserting a full length or a part of HGFA cDNA coding for the HGFAprecursor disclosed in Japanese Patent Laid-open Publication No.6-153946 into a suitable vector, introducing the vector into amicroorganism such as Escherichia coli, an insect cell, yeast, an animalcell or an animal and performing purification operation for culturesupernatant of the transgenic cells, intracellular content, tissue orbody fluid.

It is also possible to produce a target HGFA, without using a cellsystem, by using an in vitro transcription and translation systemutilizing Rapid Translation System RTS500 (Roshe Diagnostics) or thelike.

Specifically, there can be used a method utilizing an expression vectorobtained by inserting HGFA cDNA coding for 655 amino acids thatcorrespond to the full length of inactive HGFA precursor including thesignal sequence disclosed in Japanese Patent Laid-open Publication No.6-153946 into an animal cell expression vector downstream from apromoter, a method utilizing an animal cell expression vector obtainedby ligating a suitable signal sequence and cDNA coding for theC-terminus side portion of the HGFA precursor from valine at a positionof 373, which is counted from the translation initiation amino acid, asdisclosed in Japanese Patent Laid-open Publication 6-153966, and amethod utilizing an animal cell expression vector obtained by ligating asuitable signal sequence and cDNA coding for a region having an abilityto exert the HGFA activity. HGFA that is a recombinant protein can beobtained by introducing any of these expression vectors into an animalcell, then selecting a cell expressing the HGFA cDNA, and purifying HGFAof interest from culture supernatant of the cell.

HGFA that is a recombinant protein obtained by such a method isgenerally inactive HGFA. This inactive HGFA can be activated by addingsuitable amounts of thrombin and dextran sulfate or kallikrein andthrombin to the inactive HGFA referring to the method of Shimomura etal. (J. Biol. Chem., 268:22927-22932 (1993)). Purity of HGFA activatedby this treatment can be increased through purification by gelfiltration or affinity chromatography utilizing HPLC.

Not only purified active HGFA and inactive HGFA of high purity areimportant as immunogens, but also they can serve as extremely importantmaterials in case of selection of active HGFA specific polyclonalantibodies by affinity purification or screening of active HGFA specificmonoclonal antibodies.

As a result of detailed study of the inventors of the present invention,it was found that purified inactive HGFA was extremely unstable and itmight be activated by contamination of a small amount of impurities. Ifsuch inactive HGFA contaminated with active HGFA is used, or conversely,if active HGFA contaminated with inactive HGFA is used, it would bedifficult to obtain an active HGFA specific polyclonal antibody or anactive HGFA specific monoclonal antibody. Then, the inventors of thepresent invention investigated various protease inhibitors as aninhibitor for preventing the artificial activation at the time ofpreparing inactive HGFA. As a result, they found that it was effectiveto add argatroban (Mitsubishi-Tokyo Pharmaceuticals), which is aselective thrombin inhibitor, to HGFA. That is, if argatroban is addedto inactive HGFA upon purification of the inactive HGFA or screening ofmonoclonal antibodies, it is possible to prevent the artificialactivation thereof and contamination with active HGFA. Active HGFAprepared in such a manner is suitable as an antigen for immunization forobtaining an active HGFA specific antibody, and it can also be used as amaterial for selecting and purifying an antibody that recognizes activeHGFA but does not substantially recognize inactive HGFA.

On the other hand, inactive HGFA is suitable as an antigen forimmunization for obtaining an antibody that recognizes inactive HGFA butdoes not substantially recognize active HGFA, and it can be used as amaterial for selecting, absorbing or removing antibodies other than theantibody that recognizes active HGFA and does not substantiallyrecognize inactive HGFA.

It is possible to use a peptide located at a site showing difference ofprimary structure sequence (amino acid sequence) or difference ofconformation between active HGFA and inactive HGFA as an antigen forimmunization required for obtaining an active HGFA specific antibody oran antigen for screening. For example, it is possible to use a peptideat a site that is exposed only on the protein surface of active HGFA,but is not exposed on the protein surface of inactive HGFA. Suchconformation of proteins can generally be predicted by those skilled inthe art, and three-dimensional conformational structures diagrammed byusing a computer program based on the amino acid sequences of activeHGFA and inactive HGFA are available. By comparison of the bothconformations, it is possible to find out a region that serves as abinding site for an antibody showing specific reactivity to active HGFA(antigenic site) and use a peptide obtained by synthesizing a partialamino acid sequence of that region as an immunogen.

Further, it is also possible to find out a region that serves as a siteshowing a difference between active HGFA and inactive HGFA forhydrophilicity or hydrophobicity, difference in results of secondarystructure analysis based on the Cho-Fasman method, the Robson method orthe like or difference in antigenicity based on the information of theamino acid sequence of HGFA by using protein structure analysis softwaresuch as GENETYX (SOFTWARE DEVELOPMENT CO., LTD), and use a peptideobtained by synthesizing a partial amino acid sequence of that region asan immunogen or screening antigen. For example, active HGFA has an aminoacid sequence obtained by limited proteolysis of the HGFA precursorprotein between arginine at a position of the 407 and isoleucine at aposition of 408, which are counted from the translation initiation aminoacid. On the other hand, inactive HGFA has an amino acid sequence inwhich these 407th arginine and 408th isoleucine are connected.Therefore, as an amino acid sequence that does not exist in inactiveHGFA but exists only in inactive HGFA, a peptide containing the 407tharginine as the C-terminus and a sequence of several amino acidsexisting on its N-terminus side can be utilized. As example of such apeptide, the followings can be selected. The amino acid sequences ofSequences 2 to 12 correspond to sequences formed by deleting amino acidresidues from the amino acid sequence of Sequence 1 (SEQ ID NO: 1) oneby one from the N-terminus side. Sequence 1: GRRHKKRTFLRPR (SEQ. ID.NO.: 1) Sequence 2: RRHKKRTFLRPR (SEQ. ID. NO.: 4) Sequence 3:RHKKRTFLRPR (SEQ. ID. NO.: 5) Sequence 4: HKKRTFLRPR (SEQ. ID. NO.: 6)Sequence 5: KKRTFLRPR (SEQ. ID. NO.: 7) Sequence 6: KRTFLRPR (SEQ. ID.NO.: 8) Sequence 7: RTFLRPR (SEQ. ID. NO.: 9) Sequence 8: TFLRPR (SEQ.ID. NO.: 10) Sequence 9: FLRPR (SEQ. ID. NO.: 11) Sequence 10: LRPR(SEQ. ID. NO.: 12) Sequence 11: RPR Sequence 12: PR

Further, as an amino acid sequence that does not exist in inactive HGFAbut exists only in active HGFA, a peptide containing the isoleucine at aposition of 408 as the N-terminus and a sequence of several amino acidsexisting on its C-terminus side, the followings can be selected. Theamino acid sequences of Sequences 14 to 24 correspond to sequencesformed by deleting amino acid residues from the amino acid sequence ofSequence 13 (SEQ ID NO: 2) one by one from the C-terminus side. Sequence13: IIGGSSSLPGSHP (SEQ. ID. NO.: 2) Sequence 14: IIGGSSSLPGSH (SEQ. ID.NO.: 13) Sequence 15: IIGGSSSLPGS (SEQ. ID. NO.: 14) Sequence 16:IIGGSSSLPG (SEQ. ID. NO.: 15) Sequence 17: IIGGSSSLP (SEQ. ID. NO.: 16)Sequence 18: IIGGSSSL (SEQ. ID. NO.: 17) Sequence 19: IIGGSSS (SEQ. ID.NO.: 18) Sequence 20: IIGGSS (SEQ. ID. NO.: 19) Sequence 21: IIGGS (SEQ.ID. NO.: 20) Sequence 22: IIGG (SEQ. ID. NO.: 21) Sequence 23: IIGSequence 24: II

Further, as an amino acid sequence that does not exist in active HGFAbut exists only in inactive HGFA, a peptide containing an amino acidsequence in which the arginine at a position of 407 and isoleucine at aposition of 408 (between the positions of 13 and 14 of Sequence 25) ofthe HGFA precursor, which are counted from the translation initiationamino acid, are connected, such as Sequence 25 (SEQ ID NO: 3), can beselected. Sequence 25: GRRHKKRTFLRPRIIGGSSSLPGSHP (SEQ. ID. NO.: 3)

When these peptides are used as antigens for immunization, it isgenerally desirable to use those peptide bound to a protein or polymersuch as KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin) andOVA (ovalbumin) as a carrier as an antigen for immunization. Forexample, those consisting one of the peptides of Sequences 1 to 12 addedwith cysteine at the N-terminus, those consisting one of the peptides ofSequences 13 to 24 added with cysteine at the C-terminus, and thoseconsisting the peptide of Sequence 25 added with cysteine at theN-terminus or C-terminus can be synthesized, bound to Imject MaleimideActivated Carrier Proteins (PIERCE), and use as antigens forimmunization.

Moreover, it is also possible to use a fusion protein obtained byligating cDNA corresponding any one of Sequences 1 to 25 and cDNA for aprotein that serves as the carrier, expressing a fusion proteinconsisting of the peptides encoded by the both from it in various cellsusing genetic engineering techniques, and purifying the fusion protein.A peptide containing any one of Sequences 1 to 24 is suitable as anantigen for immunization for obtaining an active HGFA specific antibody,and can also be used as a material for selecting and purifying an activeHGFA specific antibody. On the other hand, a peptide like Sequence 25 issuitable as an antigen for immunization for obtaining an antibody thatrecognizes inactive HGFA but does not substantially recognize activeHGFA, and can be used as a material for absorbing or removing antibodiesother than active HGFA specific antibody.

<2> Production of Monoclonal Antibody Recognizing Active HGFA and NotSubstantially Recognizing Inactive HGFA

In order to obtain an active HGFA specific monoclonal antibody, ausually performed immunological method can be carried out by using theaforementioned active HGFA as an antigen for immunization. For example,active 98 kDa HGFA, active 36 kDa HGFA, or a mixture of the both can beused.

Further, a peptide containing any one of Sequences 1 to 24 is fused to acarrier by the method described above and one or more kinds of such asubstance can be mixed and used as an antigen for immunization.Furthermore, it is also possible to use a peptide containing a specificantigenic site for active HGFA.

The animal used for immunization is not particularly limited, and any ofrabbit, goat, sheep, mouse, rat, guinea pig, fowl and so forth can beused. The antigen for immunization is sufficiently mixed with completeFreund's adjuvant, incomplete Freund's adjuvant or the like, and theninoculated to an animal subcutaneously, intramuscularly orintraperitoneally. The inoculation is performed every 2 weeks to 5 weeksand continued until antibody titer of the immunized animal with respectto the inoculated antigen sufficiently rises. Then, only the antigen isadministered to the immunized animal by intravenous injection, after 3days, spleen or lymph node considered to contain antibody-producingcells are collected, and the spleen cell or lymph cell is fused to atumor cell. Then, an antibody-producing cell immortalized by the cellfusion (hybridoma) is isolated. While it is desirable that the tumorcell used herein should be derived from the same species as theimmunized animal from which the spleen cell or lymph cell is prepared,it may be heterozoic tumor cell.

As examples of the tumor cell, myeloma cells such as p3 (p3/x63-Ag8),P3U1, NS-1, MPC-11, SP 2/0, FO, x63.6.5.3, S194 and R210 are used. Thecell fusion can be performed by a method generally performed, and it canbe performed according to, for example, the description of “MonoclonalAntibody Experimental Manual” (Kodansha Scientific, 1987). The cellfusion can be performed by adding a cell fusion promoting agent to afusion medium in which cells to be fused are suspended. Examples of thecell fusion promoting agent include Sendai virus, polyethylene glycolhaving an average molecular weight of 1000-6000 and so forth. In thiscase, in order to further enhance fusion efficiency, auxiliary materialssuch as dimethyl sulfoxide, cytokines such as IL-6 and so forth can alsobe added to the fusion medium. As for the mixing ratio of the tumorcells to the immunized spleen cells or lymph cells, for example, thespleen cells or lymph cell can be used in an approximately equivalentamount to 10-fold amount with respect to the tumor cells.

As the aforementioned fusion medium, various usual media such as ERDFmedium, RPMI-1640 medium and MEM medium can be used, and it is usuallydesirable to eliminate blood serum such as fetal bovine serum (FBS) fromthe medium during the fusion. The fusion is performed by sufficientlymixing predetermined amounts of the spleen cells or lymph cellssubjected to the aforementioned immunization and the tumor cells in theaforementioned medium, adding a polyethylene glycol solution warmed toabout 37° C. beforehand in an amount of from about 20% to 50%, andallowing them to react preferably at 30° C. to 37° C. for about 1 minuteto 10 minutes. Thereafter, there are repeated operations of occasionallyadding a suitable medium, centrifuging the medium and removing thesupernatant.

A target hybridoma is cultured in a usual selective medium, for example,HAT medium (medium containing hypoxanthine, aminopterin and thymidine).The culture in this HAT medium may be performed for a period sufficientfor cells other than the hybridoma (not fused cells etc.) to be killed,usually several days to several weeks. As for acquisition of an activeHGFA specific monoclonal antibody, the most technically important pointis the screening thereof. The screening of a hybridoma producing activeHGFA specific monoclonal antibody is enabled by using the aforementionedmaterials such as active HGFA and inactive HGFA and performing analysesthorough various immunochemical methods. For example, a target hybridomacan be selected by using active HGFA or inactive HGFA as an antigen forscreening and analyzing the binding of the antigen for screening with amonoclonal antibody secreted in the culture supernatant of the hybridomabased on an enzyme immunoassay such as ELISA, Western blotting or thelike.

Specifically, active HGFA is adhered to a screening plate or the like,blocked with BSA or the like, and added with the aforementioned culturesupernatant of hybridoma to select hybridomas secreting antibodies thatrecognize active HGFA. The selected hybridomas were added to inactiveHGFA adhered to a screening plate or the like and blocked with BSA orthe like to further select hybridomas secreting antibodies that do notrecognize this inactive HGFA. For example, the culture supernatant ofhybridoma subjected to selection is added to a plate for ELISA adheredwith active HGFA or inactive HGFA and allowed to react, and aftersufficient washing operation, the plate was added with labeledanti-mouse IgG polyclonal antibodies to further perform a reaction.After washing operation, the label is detected to select a hybridomathat provides a culture supernatant that shows reactivity to the plateadhered with active HGFA, but showing no reactivity to the plate adheredwith inactive HGFA. As the label, there are used various enzymes,fluorescent substances, chemiluminescent substances, radioactiveisotopes, biotin, avidin and so forth, which will be described later.

By the above screening, there can be obtained a hybridoma that producinga monoclonal antibody that recognizes active HGFA but does notsubstantially recognize inactive HGFA. Further, in the screening of sucha hybridoma, an event that an antibody reacts with a peptide containingany one of the aforementioned Sequences 1 to 24 but does not react witha peptide containing Sequence 25 or the like can be used as an index. Asfor an antibody produced by a hybridoma selected in such a manner, it ispreferable to further confirm that the antibody reacts with active HGFAbut does not react with inactive HGFA.

The reactivity of the aforementioned antibody can be confirmed bymeasuring the dissociation constant for active HGFA or inactive HGFA.The dissociation constant of the antibody of the present invention withrespect to active HGFA is preferably 1×10⁻⁸ M or less, more preferably1×10⁻⁹ M or less. The dissociation constant with respect to inactiveHGFA is preferably 1×10⁻⁵ M or more.

The obtained hybridoma can be cloned by the limiting dilution method toobtain a hybridoma clone producing a single kind of monoclonal antibody.This hybridoma clone is cultured in a medium supplemented with about 1to 5% FBS from which bovine antibodies (IgG) contained in the FBS areeliminated beforehand or a medium for serum free culture, and theobtained culture supernatant is used as a raw material for purificationof the target monoclonal antibody. Further, it is also possible totransfer the obtained hybridoma clone into abdominal cavity of Balb/Cmouse or Balb/c (nu/nu) mouse administered with pristane beforehand andextract ascites containing monoclonal antibodies at a high concentration10 to 14 days later to use it as a raw material for purification of thetarget monoclonal antibody. As the method of purifying monoclonalantibodies, usual method for purifying immunoglobulin can be used, andit can be performed by, for example, ammonium sulfate fractionation,polyethylene fractionation, ethanol fractionation, anion exchangechromatography, affinity chromatography utilizing protein A or protein Gand so forth.

<3> Production of Polyclonal Antibody That Recognizes Active HGFA andDoes Not Substantially Recognize Inactive HGFA

An active HGFA specific polyclonal antibody can be obtained byimmunization with active HGFA or inactive HGFA as an antigen forimmunization and purification of an antibody that recognizes active HGFAand does not substantially recognize inactive HGFA from polyclonalantibodies derived from the obtained immunized animal.

Further, as the antigen for immunization for obtaining polyclonalantibodies, the aforementioned fused substance of a peptide containing aspecific antigenic site for inactive HGFA and a carrier can be used. Forexample, one or more kinds of substance consisting of a peptidecontaining any one of Sequences 1 to 25 fused to a carrier protein or apolymer compound may be mixed and used as the antigen for immunization.The animal used for the immunization is not particularly limited, andany of rabbit, goat, sheep, mouse, rat, guinea pig, fowl and so forthcan be used. The antigen for immunization is sufficiently mixed withcomplete Freund's adjuvant, incomplete Freund's adjuvant or the like,and then inoculated to an animal subcutaneously, intramuscularly orintraperitoneally. The inoculation is performed every 2 weeks to 5 weeksand continued until antibody titer of the immunized animal with respectto the inoculated antigen sufficiently rises. Then, only the antigen isadministered to the immunized animal by intravenous injection, and after3 days to 5 days, anti-serum is collected.

As the method of purifying polyclonal antibodies from the obtainedanti-serum, a usual method for purifying immunoglobulin can be used, andit can be performed by, for example, ammonium sulfate fractionation,polyethylene fractionation, ethanol fractionation, anion exchangechromatography, affinity chromatography utilizing protein A or protein Gand so forth.

The purification operation for obtaining an active HGFA specificpolyclonal antibody can be performed by any method so long as the methodcan fractionate or purify a polyclonal antibody that recognizes activeHGFA and does not substantially recognize inactive HGFA, and examples ofthe method include, for example, ion exchange chromatography,hydrophobic chromatography, molecular sieve chromatography, reversephase chromatography, hydroxyapatite chromatography, affinitychromatography, gel electrophoresis, immunoelectrophoresis and so forth.As one of specific methods, there can be mentioned affinity columnchromatography using a resin on which active HGFA or inactive HGFA isimmobilized. For example, by using the polyclonal antibodies obtained bythe aforementioned method as a raw material, affinity chromatographyusing a resin on which inactive HGFA is immobilized is performed. Bythis method, polyclonal antibodies that are not bound to inactive HGFA,which exist in a non-adsorbed fraction, are collected. Then, thisnon-adsorbed fraction is used as a raw material to perform affinitychromatography utilizing a resin on which active HGFA is immobilized andthereby collect polyclonal antibodies that are bound to active HGFA,which exist in an adsorbed fraction.

By these methods, an active HGFA specific polyclonal antibody can beobtained. Further, for another method of the affinity chromatography, itis also possible to use a peptide containing an amino acid sequenceconsidered to be an antigen site specific for active HGFA. For example,it is possible to utilize affinity chromatography using a resin on whichone or more kinds of Sequences 1 to 25 are immobilized. For example, aresin on which a peptide containing any of Sequence 1 to 24 isimmobilized can be used as an immobilization carrier for affinitychromatography used for purification of an active HGFA specificpolyclonal antibody. On the other hand, a resin on which a peptide ofSequence 25 or the like is immobilized can be used as an immobilizationcarrier for affinity chromatography used for adsorbing or removingantibodies other than the active HGFA specific antibody.

<4>Method for Specifically Measuring Active HGFA

A method for specifically measuring active HGFA means a methodcomprising a process of allowing an active HGFA specific antibody toreact with active HGFA. Therefore, this method is a method characterizedby measuring active HGFA and not substantially measuring inactive HGFA.This method can be used for various diagnostic methods, measurementmethods and assay methods, in which active HGFA in a biosample isqualitatively or quantitatively measured by using an active HGFAspecific antibody of the present invention. The methods are notparticularly limited so long as the methods are for a purpose ofdetecting active HGFA. Examples of the methods include, for example,tissue staining methods and immunoprecipitation methods for specificallydetecting active HGFA, competitive binding assay methods forspecifically measuring active HGFA, direct or indirect sandwich assaymethods, double antibody sandwich assay methods and so forth. Further,examples of detection methods include enzyme immunoassays,radioimmunoassays, fluorescent immunoassays, chemiluminescenceimmunoassays, immunoblotting methods, immunochromatography methods,latex agglutination methods and so forth.

Examples of applications of immunoblotting include those utilizingactive HGFA specific antibodies immobilized on a microarray or chip. Itis also possible to label active HGFA specific antibodies withfluorescent label and detect its interaction with active HGFA using thefluorescence depolarization method or the fluorescence correlationvariance method. It is also possible to measure the interaction of anactive HGFA specific antibody with active HGFA by using a surfaceplasmon resonance apparatus. For example, it is possible toquantitatively measure active HGFA in a biological component by flowinga sample of the biological component containing active HGFA in a surfaceplasmon resonance apparatus provided with a sensor chip on which theantibodies are bonded and tracing variation of the response signal withtime.

The antibody used in a method for specifically measuring active HGFA,for example, an active HGFA specific antibody, may be used as it is, orused as an antibody in the form of Fab, which is obtained by theconventional papain treatment, or in the form of F(ab′)₂ or F(ab′),which is obtained by the pepsin treatment. Further, an antibody fragmentthat has a property of recognizing active HGFA and not substantiallyrecognizing inactive HGFA also fall within the scope of the presentinvention. Examples of such a fragment include, for example, a fragmentcontaining a complementation determination region (CDR) or ahypervariable region in variable domains of both of heavy chain andlight chain of an active HGFA specific antibody and so forth.

The double antibody sandwich assay method for quantitatively determiningactive HGFA in a biological component may be, for example,

a method for specifically measuring active HGFA, which comprises (1) astep of allowing a reagent comprising one or more kinds of active HGFAspecific antibodies to react with active HGFA in a specimen to form animmunological reaction product, (2) a step of separating theimmunological reaction product and then allowing it to react withlabeled antibodies that recognize HGFA contained in the immunologicalreaction product, and (3) a step of measuring the labeled antibodiesbound to the immunological reaction product, or

a method for specifically measuring active HGFA, which comprises (1) astep of allowing a reagent comprising active HGFA and one or more kindsof active HGFA specific antibodies as primary antibodies to react withactive HGFA in a specimen to form an immunological reaction product, (2)a step of separating the immunological reaction product and thenallowing it to react with secondary antibodies that recognize HGFAcontained in the immunological reaction product to form an immunologicalreaction product, (3) a step of separating the immunological reactionproduct and then allowing it to react with labeled antibodies thatrecognize the secondary antibodies contained in the immunologicalreaction product, and (4) a step of measuring the labeled antibodiesbound to the immunological reaction product.

Specifically, active HGFA specific polyclonal antibodies or active HGFAspecific monoclonal antibodies are immobilized in a conventional manneras primary antibodies on a solid phase such as microtiter wells andmagnetic microbeads. Then, excessive protein bonding sites on the solidphase surface are blocked with bovine serum albumin, skim milk, gelatinor the like. Then, a biological component containing active HGFA isadded to form an immunological reaction product on the solid phase andthen the solid phase is washed. Subsequently, labeled polyclonalantibodies or labeled monoclonal antibodies that recognize HGFA areadded as secondary antibodies and allowed to react. In this case, whenmonoclonal antibodies are used as the primary antibodies, labeled activeHGFA specific monoclonal antibodies having an epitope different fromthat of the primary antibodies may be used as the secondary antibodies.Further, after washing, the amount of labeled antibodies can be measuredto determine the amount of active HGFA in the biological component.

The label of the polyclonal antibodies or monoclonal antibodies usedhere may be an enzyme such as alkaline phosphatase, horseradishperoxidase, β-galactosidase, urease and glucose oxidase, or afluorescent substance such as fluorescein derivatives and rhodaminederivatives. Moreover, the label may be a chemiluminescent substancesuch as acridinium esters or a radioactive isotope such as ¹²⁵I, ³H, ¹⁴Cand ³²P. That is, the present invention include quantitativelydetermining an amount of active HGFA in a biological component using amethod of measuring luminescence, fluorescence, chemiluminescence,electrochemical luminescence or radioactivity. The present inventionalso includes a method comprising biotinylating the secondary antibodiesand detecting alkaline phosphatase, horseradish peroxidase,β-galactosidase, urease, glucose oxidase, fluorescein derivative,rhodamine derivative, a chemiluminescent substance such as acridiniumesters or a radioactive isotope such as ¹²⁵I, ³H, ¹⁴C and ³²P forming acomplex with avidin.

<5> Kit for Specifically Measuring or Staining Active HGFA

An active HGFA specific measurement kit or an active HGFA specificstaining kit is a kit used for diagnosing a disease characterized bymeasuring or detecting active HGFA. The term “specific” used here meansthat, when active HGFA is measured or detected, inactive HGFA does notaffect a measured value or detection value of active HGFA. It was foundby the present invention for the first time that, by measuring activeHGFA, diagnosis or prediction of a patient having a pathologicalcondition, for example, a patient with organ derangement such asglomerular nephritis, nephritis, hepatitis, pancreatitis, pneumonitis,enteritis and gastritis, a cancer patient, and a patient with thrombosissuch as angina pectoris, myocardial infarction and cerebral infarction.Therefore, by measuring or detecting active HGFA, various diseases suchas those mentioned above can be detected. In the present invention,materials constituting the kit or methods for which the kit is used arenot particularly limited so long as the kit is an active HGFA specificmeasurement kit for the purpose of diagnosing a disease.

Specifically, examples of the kit includes those used for diagnosing adisease by measuring or detecting active HGFA using electrophoresis,HPLC, various column chromatography techniques, various arrays andchips, surface plasmon resonance apparatus and so forth. Morespecifically, there can be mentioned a kit for measuring or detectingactive HGFA by an immunological method utilizing an antibody. As theantibody, at least one or more kinds of the aforementioned antibodiesthat recognize active HGFA and do not substantially recognize inactiveHGFA are used.

For example, when the kit of the present invention is based on thedouble antibody sandwich assay method, the kit may be

a kit for specifically measuring active HGFA, which is used for a methodcomprising (1) a step of allowing a reagent comprising active HGFA andone or more kinds of active HGFA specific antibodies to react withactive HGFA in a specimen to form an immunological reaction product, (2)a step of separating the immunological reaction product and thenallowing it to react with labeled antibodies that recognize HGFAcontained in the immunological reaction product, and (3) a step ofmeasuring the labeled antibodies bound to the immunological reactionproduct, or

a kit for specifically measuring active HGFA, which is used for a methodcomprising (1) a step of allowing a reagent comprising one or more kindsof active HGFA specific antibodies as primary antibodies to react withactive HGFA in a specimen to form an immunological reaction product, (2)a step of separating the immunological reaction product and thenallowing it to react with secondary antibodies that recognize HGFAcontained in the immunological reaction product to form an immunologicalreaction product, (3) a step of separating the immunological reactionproduct and then allowing it to react with labeled antibodies thatrecognize the secondary antibodies contained in the immunologicalreaction product, and (4) a step of measuring the labeled antibodiesbound to the immunological reaction product.

This kit contains at least an active HGFA specific monoclonal antibodyor an active HGFA specific polyclonal antibody, and may further containcomponents required for the process of detecting or measuring activeHGFA. Examples of the components include active HGFA or inactive HGFA asa standard protein, an enzyme, a substrate and so forth. The monoclonalantibody or polyclonal antibody contained in the kit may be an antibodylabeled with an enzyme or the like, or the kit may contain a labeledantibody that recognizes the aforementioned antibody. Further, the kitmay contain various buffers, solutions for diluting antigen, solutionsfor diluting reaction mixture, substrate solutions, solutions forstopping reaction and so forth. The kit may contain a container attachedwith a label and enclosing materials required for detection andquantitative determination of active HGFA. Examples of a suitablecontainer include containers composed of glass or various plasticmaterials such as polypropylene, polystyrene, polycarbonate, nylon andTeflon. The kit preferably include an instruction describing a method ofdetecting or measuring active HGFA together with the aforementionedmaterials required for detecting or measuring active HGFA and acontainer.

<6> Active HGFA Specific Antibody Concerning Human Disease and Methodfor Using the Same

By using the method and kit utilizing the active HGFA specific antibodyaccording to the present invention, active HGFA in a biologicalcomponent collected from a patient in a pathological condition can bedetected or even quantitatively measured. The biomaterial for whichactive HGFA is detected is not particularly limited, and any of tissues,blood serum, plasma, urine, serous fluid, spinal fluid, extract oftissue and so forth can be used by subjecting them to a suitablepretreatment. By detecting or quantitatively determining active HGFAexisting in a biomaterial collected from a patient in a pathologicalcondition, the disease can be diagnosed and predicted or progress of thedisease can be determined. Examples of the disease include organderangement such as glomerular nephritis, nephritis, hepatitis,pancreatitis, pneumonitis, enteritis and gastritis, cancer, thrombosissuch as angina pectoris, myocardial infarction and cerebral infarctionand so forth.

Examples of nephritis, in particular, include mesangial proliferativenephropathy, IgA nephritis, membranoproliferative glomerulonephritis,membranous nephropathy, focal glomerular sclerosis, acute renal failure,poststreptococcal acute glomerulonephritis, chronic and acuteinterstitial nephritis, nephrotic syndrome and so forth. Examples ofangina pectoris and myocardial infarction include stable exertionalangina pectoris, unstable angina pectoris, acute myocardial infarction,old myocardial infarction and stable angina pectoris, and it is alsopossible to know pathological condition or prognosis of patientssubjected to coronary intervention, trans-esophageal echocardiography,lower extremity artery bypass operation or aortic balloon pumping,patients with acute aortic dissection and so forth.

Moreover, since an active HGFA specific antibody can be expected to havean effect of specifically inhibiting the activity of active HGFA, it isexpected to be used as a drug for therapeutic treatment of a diseasecaused by active HGFA. For example, active HGFA has a property of actingon inactive HGF to activate it. Therefore, an antibody that inhibits theactivity of active HGFA suppresses increase of amount of active HGFemerging in living bodies, and it can be used as therapeutic orprophylactic drug for diseases considered to be caused by active HGF(WO96/38557; Genentech Incorporated, “Molecular Medicine of HGF”,Medical Review, 1998), for example, cancers such as stomach cancer, lungcancer, colon cancer, spleen cancer and liver cancer as well asmetastases thereof, various kinds of nephritis such as glomerularnephritis and so forth.

Existence of mouse monoclonal antibody that inhibits the activity ofactive HGFA (monoclonal antibody P1-4) was disclosed by Miyazawa et al.(J. Biol. Chem., 271:3615-3618 (1996)), and it was elucidated by thestudy of the inventors of the present invention that this antibody P4-1reacted to both of active HGFA and inactive HGFA. Since inactive HGFAabundantly exists in living bodies, it is considered that an extremelylarge amount of P4-1 antibodies are required for suppressing theactivity of active HGFA. On the other hand, since the active typespecific antibody of the present invention is presumed to be an antibodythat inhibits the activity of active HGFA, it is considered that it willexert superior effect as a therapeutic drug for the aforementioneddiseases with a small amount.

The antibody used for this purpose is preferably humanized by using agenetic engineering technique. Humanization of antibody can be performedby a method well known to those skilled in the art, for example, onedisclosed in International Patent Publication in Japanese (Tokuhyo) No.11-506327 and so forth.

<7> Blood Collection Method and Blood Collection Tube for MeasuringActive HGFA

While the biological component for detection or measurement of activeHGFA is not particularly limited, it is usually blood or a fraction orprocessed product thereof, and it is desirably a biological componentthat can be collected from a vessel such as blood, blood serum, plasmasuch as citrated plasma, heparin plasma and EDTA plasma, fractions orprocessed products thereof, or urine that can be easily collected.During collection and storage of a biological component, in particular,a manipulation for preventing inactive HGFA contained in the biologicalsample from being artificially converted into active HGFA is required.For example, it is desirable that collected blood serum or plasma, urineand so forth should immediately be placed under a low temperature suchas under ice cooling.

Further, in order to quickly collect blood, blood serum, citratedplasma, heparin plasma or EDTA plasma from a vessel, it is desirable touse a blood collection tube. In particular, blood for use in detectionor measurement of active HGFA is preferably in a state of plasma,especially citrated plasma. Further, a method of adding various proteaseinhibitors to the biological component for detection or measurement ofactive HGFA is also preferred. For example, if argatroban, which is aselective thrombin inhibitor, is added to a collected biosample in orderto prevent inactive HGFA contained in a biological component from beingartificially converted into active HGFA during collection or storage ofthe biological component, good result can be obtained. It isparticularly preferable to use a tube added beforehand with argatrobanfor collection of blood, blood serum, citrated plasma, heparin plasma orEDTA plasma.

<8> Method for Screening of Protease Inhibitor for Active HGFA

By using an antibody that recognizes active HGFA, which does notsubstantially recognize inactive HGFA and does not recognize a complexof active HGFA and a protease inhibitor, a protease inhibitor that actson active HGFA can be screened.

An antibody having the aforementioned property can be obtained byfurther selecting an antibody that does not recognize a complex ofactive HGFA and a protease inhibitor from active HGFA specificantibodies.

The aforementioned screening method specifically comprises, for example,(1) a step of mixing active HGFA and a candidate compound for proteaseinhibitor and allowing them to react, (2) a step of adding a monoclonalantibody that recognizes active HGFA but does not substantiallyrecognize inactive HGFA and does not recognize a complex of active HGFAand a protease inhibitor to the mixture of active HGFA and the candidatecompound for protease inhibitor, and (3) a step of separating animmnological reaction product and then measuring decrease of amount ofthe immunological reaction product consisting of the complex of activeHGFA and the candidate compound for protease inhibitor and the antibody.

As the method of detecting the decrease of the amount of thisimmunological reaction product, there can be used enzyme immunoassays,radioimmunoassays, fluorescent immunoassays, chemiluminescenceimmunoassays, electrochemical luminescence immunoassays, immunoblottingmethods, immunochromatography methods, latex agglutination methods,microarray methods that are applications of immunoblotting, and methodsutilizing the fluorescence depolarization method, the fluorescencecorrelation variance method or a surface plasmon resonance apparatus.For example, it is possible to screen a protease inhibitor by flowing asample of biosample in a surface plasmon resonance apparatus providedwith a sensor chip on which antibodies that recognize active HGFA but donot substantially recognize inactive HGFA and do not recognize a complexof active HGFA and a protease inhibitor are bonded and tracing variationof the response signal with time.

The antibody used in this method may be used as it is, or used as anantibody in the form of Fab, which is obtained by the conventionalpapain treatment, or in the form of F(ab′)₂ or F(ab′), which is obtainedby the pepsin treatment. Further, an antibody fragment that has theproperty of the antibody used in this method can also be used.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained more specifically with referenceto the following examples. However, the scope of the present inventionis not limited to these examples.

EXAMPLE 1 Preparation of Inactive HGFA and Active HGFA

Inactive HGFA was prepared by using HGFA cDNA coding for the HGFAprecursor described in Japanese Patent Laid-open Publication No.6-153946 according to the method described in Japanese Patent Laid-openPublication No. 6-153966 to produce recombinant inactive HGFA. That is,the HGFA cDNA coding for the full length of the inactive HGFA precursor,655 amino acids, was inserted into pcDNA3.1 (Invitrogen), which is ananimal cell expression vector, downstream from a CMV promoter in aconventional manner.

The obtained expression vector was introduced into a CHO cell, which isan animal cell strain derived from a Chinese hamster ovary cell, byusing a Transfectam (BioSepra) according to the attached instruction.Then, CHO cells expressing HGFA cDNA were selected by utilizing aproperty of a neomycin resistant gene existing on the introducedexpression vector. That is, cells that could grow in an ERDF medium(Kyokuto Seiyaku) containing 400 μg/ml neomycin and 5% fetal bovineserum under 5% CO₂ at 37° C. were selected. Further, the selected CHOcells expressing the HGFA cDNA were cultured in an ERDF medium (KyokutoSeiyaku) containing 100 μM nafamostat mesylate, 400 μg/ml neomycin and5% fetal bovine serum for about 10 days to obtain about 5 L of culturesupernatant.

The obtained culture supernatant was filtered, added with a proteaseinhibitor containing 10 mM EDTA, 10 mM benzamidine, 100 μM nafamostatmesylate, soybean trypsin inhibitor and 1000 KIU/ml aprotinin andfurther added with 0.5 M acetate buffer (pH 4.5) to adjust the culturesupernatant to about pH 5.8. This culture supernatant was applied to asulfated Cellofine column (Seikagaku Corporation) equilibrated with 50mM acetate buffer (pH 5.5) containing 100 mM NaCl and washed with 50 mMacetate buffer (pH 5.5) containing 100 mM NaCl. Then, elution wasperformed with 50 mM sodium phosphate buffer (pH 7.5) containing 500 mMNaCl, 0.05% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonicacid) to obtain an inactive HGFA fraction.

The obtained fraction was dialyzed against 50 mM sodium phosphate buffer(pH 7.5) containing 150 mM NaCl, applied to an A6 monoclonal antibodyaffinity column (Shimomura et al., J. Biol. Chem., 268:22927-22932,1993) and eluted with 50 mM glycine-HCl buffer (pH 3.0). The buffer ofthe obtained inactive HGFA fraction was replaced with 10 mM sodiumphosphate buffer (pH 7.3) containing 100 mM NaCl and 0.05% CHAPS. A partof the fraction was added with argatroban, which is a proteaseinhibitor, to a final concentration of 40 μM and stored as inactive HGFAat −40° C. On the other hand, inactive HGFA not added with argatrobanwas added with plasma kallikrein and thrombin in an amount of 1/100 inweight ratio to the HGFA and allowed to sufficiently react at 37° C. toobtain active 36 kDa HGFA.

Further, the inactive HGFA not added with argatroban was added withthrombin in an amount of 1/100 in weight ratio to the HGFA and 10 μg/mldextran sulfate and allowed to react at 37° C. for 20 minutes to obtainactive 98 kDa HGFA. Each active HGFA fraction was purified again byusing an A6 monoclonal antibody affinity column, subjected to HPLC usingan Asahipak GS520HQ column (Showa Denko) equilibrated with 10 mM sodiumphosphate buffer (pH 7.3) containing 100 mM NaCl and 0.05% CHAPS andstored as active 36 kDa HGFA and active 98 kDa HGFA at −40° C.

EXAMPLE 2 Preparation of ELISA Plate for Screening Active HGFA SpecificMonoclonal Antibody

The active 36 kDa HGFA or active 98 kDa HGFA prepared in Example 1 wasdiluted with hydrogenphosphate buffered physiological saline (PBS(-)) toa final concentration of 1 μg/ml as an antigen for screening hybridoma,and 100 μl of the solution was added to each well of a 96-well plate andstored at 4° C. for 24 hours so that each antigen should be adsorbed onthe 96-well plate. The solution was removed from this antigen-adsorbedplate, and then 250 μl of PBS(-) containing 5% bovine serum albumin(hereafter, abbreviated as “BSA”) was added to each well and left at 4°C. overnight (about 12 hours) or at 37° C. for 2 hours or longer toblock the plate. Then, the plate was stored as an ELISA plate forscreening active HGFA at 4° C.

Separately, the inactive HGFA prepared in Example 1 was diluted withPBS(-) to a final concentration of 1 μg/ml as an antigen for screeninghybridoma. Then, 100 μl of the solution was added to each well of a96-well plate and stored at 4° C. for 24 hours so that the antigenshould be adsorbed on the 96-well plate. The solution was removed fromthe antigen-adsorbed plate, and then 250 μl of PBS(-) containing 5%bovine serum albumin (hereafter, abbreviated as “BSA”) was added to eachwell and left at 4° C. overnight (about 12 hours) or at 37° C. for 2hours or longer to block the plate. Then, the plate was stored as anELISA plate for screening inactive HGFA at 4° C. The blocking solutionin these ELISA plates was removed immediately before use.

EXAMPLE 3 Preparation of Active HGFA Specific Monoclonal Antibody

A solution containing 100 μg of the 36 kDa HGFA or 100 μg of the 98 kDaHGFA, which were prepared in Example 1, was subcutaneously andintraperitoneally administered to a Balb/c mouse with the same volume ofcomplete Freund's adjuvant 6 times with 2-week intervals. Afterproduction of antibodies in the serum of the mouse was confirmed, asolution containing 100 μg of HGFA was administered into the caudalvein. Three days later, spleen was removed and spleen cells were fusedwith myeloma cells P3U1 by using polyethylene glycol 1500 according to“Monoclonal Antibody Experimental Manual” (Kodansha Scientific, 1987),introduced into wells of 96-well plate, added with HAT medium andcultured for 14 days.

Subsequently, hybridomas producing monoclonal antibodies specific torespective active HGFA in the medium were selected. That is, culturesupernatant of a hybridoma subjected to selection was added to an ELISAplate for screening 36 kDa HGFA or active 98 kDa HGFA prepared inExample 2, and reactivity of the monoclonal antibodies existing in theculture supernatant was analyzed. In an amount of 100 μl/well of theculture supernatant of the hybridoma subjected to selection was added tothe ELISA plate for screening each activity type and allowed to react at4° C. for 2 hours or longer.

Then, the plate was sufficiently washed with a PBS(-) solutioncontaining 0.05% Tween 20 (hereafter, abbreviated as “PBST solution”),and then 100 μl of PBS(-) containing 1 μg/ml HRP (horseradishperoxidase) conjugated sheep anti-mouse IgG/Fc polyclonal antibody(DAKO) and 1% BSA was added to each well and allowed to react at roomtemperature for 1 hour. The plate was sufficiently washed with a PBSTsolution, and then a citrate-phosphate buffer (pH 5.0) containing 0.4mg/ml orthophenylenediamine (OPD, Sigma, P-9029) and a 0.015-0.03%hydrogen peroxide solution was added and allowed to react at roomtemperature for color development. Then, the reaction mixture was addedwith a 1 N H₂SO₄ solution to stop the reaction and measurement wasperformed at a measurement wavelength of 490 nm and a referencewavelength of 650 nm.

Then, by using the culture supernatant of hybridoma producing monoclonalantibodies showing reactivity to the active 36 kDa HGFA or active 98 kDaHGFA, screening was performed in the same manner on the ELISA plate forscreening inactive HGFA prepared in Example 2 to select a hybridoma thatdoes not show any reactivity to inactive HGFA. By this screening, ahybridoma producing a monoclonal antibody that recognized active 36 kDaHGFA and did not substantially recognize inactive HGFA and a monoclonalantibody that recognized active 98 kDa HGFA and did not recognizeinactive HGFA (AHGA-A, AHGA-B, AHGA-C) were obtained. Each obtainedhybridoma was subjected to four times of cloning operations by a limiteddilution method, and its culture supernatant was collected and subjectedto affinity chromatography utilizing protein A (Amersham PharmaciaBiotec) to purify monoclonal antibodies.

The hyboridoma clone AHGA-A was deposited since Oct. 19, 2001 inNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary (Tsukuba Central 6, 1-1,Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) asdeposition number of FERM BP-7779.

EXAMPLE 4 Analysis of Reaction Specificity of Active HGFA SpecificMonoclonal Antibody

Among active HGFA specific monoclonal antibodies prepared and purifiedin Example 3, reactivities of monoclonal antibodies derived fromhybridoma clones AHGA-A, AHGA-B and AHGA-C were analyzed. To thispurpose, 100 μl of PBS(-) containing about 1 μg/ml monoclonal antibodiesderived from each of hybridoma clones AHGA-A, AHGA-B and AHGA-C and 1%BSA was added to each well of the ELISA plate for screening active 36kDa HGFA or active 98 kDa HGFA or ELISA plate for screening inactiveHGFA prepared in Example 2 and allowed to react at 4° C. for 2 hours orlonger.

Subsequently, the plate was sufficiently washed with a PBS(-) solutioncontaining 0.05% Tween 20 (hereafter, abbreviated as “PBST solution”),and then 100 μl of PBS(-) containing 1 μg/ml HRP-conjugated sheepanti-mouse IgG polyclonal antibody (DAKO) and 1% BSA was added to eachwell and further allowed to react at room temperature for 1 hour. Theplate was sufficiently washed with a PBST solution, and then acitrate-phosphate buffer (pH 5.0) containing 0.4 mg/mlorthophenylenediamine (OPD, Sigma, P-9029) and a 0.015-0.03% hydrogenperoxide solution was added and allowed to react at room temperature forcolor development.

Then, the reaction mixture was added with a 1 N H₂SO₄ solution to stopthe reaction and measurement was performed at a measurement wavelengthof 490 nm and a reference wavelength of 650 nm. The measurement resultsare shown in FIG. 1. The monoclonal antibodies derived from hybridomaclones AHGA-A and AHGA-B were reactive to active 36 kDa HGFA, but notreactive to inactive HGFA. Further, those of the hybridoma clone AHGA-Cwas reactive to active 98 kDa HGFA, but not reactive to inactive HGFA(FIG. 1). Separately, reactivities to active 36 kDa HGFA and inactiveHGFA of existing monoclonal antibodies directed to HGFA (7E10, P1-4,A-1, A-6, A-23, A-32, A-51, A-75) were analyzed. As a result, theyshowed equivalent reactivities to both the active HGFA and inactive HGFAand did not have a reactivity specific to active HGFA unlike themonoclonal antibodies obtained in the present invention.

EXAMPLE 5 Measurement of Dissociation Constant of Active HGFA SpecificMonoclonal Antibody

Among active HGFA specific monoclonal antibodies prepared and purifiedin the Example 3, the dissociation constant was measured for themonoclonal antibody derived from the hybridoma clone AHGA-A. An activeHGFA immobilized plate on which active HGFA was immobilized and blockedwith BSA was added with the antibodies at different antibodyconcentrations and allowed to sufficiently react until equilibrium wasreached (2 hours or longer).

Subsequently, the plate was sufficiently washed with a PBS(-) solutioncontaining 0.05% Tween 20 (hereafter, abbreviated as “PBST solution”),and then 100 μl of PBS(-) containing 1 μg/ml of HRP (horseradishperoxidase) conjugated goat anti-mouse IgG/Fc polyclonal antibody (ICN)and 1% BSA was added to each well and further allowed to react at roomtemperature for 1 hour. The plate was sufficiently washed with a PBSTsolution, and then a citrate-phosphate buffer (pH 5.0) containing 0.4mg/ml orthophenylenediamine (OPD, Sigma, P-9029) and a 0.015-0.03%hydrogen peroxide solution were added and allowed to react at roomtemperature for color development.

Then, the reaction mixture was added with a 1 N H₂SO₄ solution to stopthe reaction and measurement was performed at a measurement wavelengthof 490 nm and a reference wavelength of 650 nm. A dissociation constantwas obtained from the measurement result by Schatchard plot. As aresult, it was 4.05×10⁻¹⁰ M. This result suggests that the antibody ofthe present invention has high affinity.

EXAMPLE 6 Preparation of Monoclonal Antibody That SpecificallyRecognizes Active HGFA and Does Not Recognize Complex of Active HGFA andProtease Inhibitor

The hybridomas producing a monoclonal antibody that specificallyrecognized active HGFA and did not recognize inactive HGFA selected inExample 3 were further screened as follows.

The blocking solution was removed from the plate for screening active 36kDa HGFA or active 98 kDa HGFA prepared in Example 2, and then 20 μl ofPBS(-) containing 200 μM nafamostat mesylate, which is a proteaseinhibitor, was added to each well and allowed to react at roomtemperature for 1 hour to form a complex of active HGFA and nafamostatmesylate. Then, 100 μl of the culture supernatant of the hybridomasubjected to selection was added to each well and allowed to react at 4°C. for 2 hours.

Subsequently, the plate was sufficiently washed with a PBS(-) solutioncontaining 0.05% Tween 20 (hereafter, abbreviated as “PBST solution”),and then 100 μl of PBS(-) containing 1 μg/ml HRP-conjugated sheepanti-mouse IgG polyclonal antibody (DAKO) and 1% BSA was added to eachwell and further allowed to react at room temperature for 1 hour. Theplate was sufficiently washed with a PBST solution, and then acitrate-phosphate buffer (pH 5.0) containing 0.4 mg/mlorthophenylenediamine (OPD, Sigma, P-9029) and a 0.015-0.03% hydrogenperoxide solution was added and allowed to react at room temperature forcolor development.

Then, the reaction mixture was added with a 1 N H₂SO₄ solution to stopthe reaction and measurement was performed at a measurement wavelengthof 490 nm and a reference wavelength of 650 nm. By this screening, amonoclonal antibody that did not recognize a complex of active 36 kDaHGFA and nafamostat mesylate was selected. A hybricoma clone producingthe selected monoclonal antibody was designated as AHGA-D. Each of theobtained hybridomas was subjected to 4 times of cloning operations by alimited dilution method, and then its culture supernatant was collectedand subjected to affinity chromatography utilizing protein A to purifythe monoclonal antibody.

EXAMPLE 7 Analysis of Reaction Specificity of Monoclonal Antibody ThatSpecifically Recognizes Active HGFA and Does Not Recognize Complex ofActive HGFA and Protease Inhibitor

The reactivity of the monoclonal antibody derived from the hybridomaclone AHGA-D selected in Example 6 was analyzed. In an amount of 20 μlof PBS(-) containing nafamostat mesylate was added at a concentration of0, 0.820, 7.40, 22.20 or 2000 μM to each well of the ELISA plate forscreening active 36 kDa HGFA prepared in Example 2 and allowed to reactat room temperature for 1 hour to form a complex of active HGFA andnafamostat mesylate. Then, 100 μl of PBS(-) containing about 1 μg/ml themonoclonal antibody derived from the hybridoma clone AHGA-D, 1% BSA andnafamostat mesylate at various concentrations (0, 0.820, 7.40, 22.20 and2000 μM) was added to each well and allowed to react at 4° C. for 2hours or longer.

Subsequently, the plate was sufficiently washed with a PBS(-) solutioncontaining 0.05% Tween 20 (hereafter, abbreviated as “PBST solution”),and then 100 μl of PBS(-) containing 1 μg/ml HRP-conjugated sheepanti-mouse IgG polyclonal antibody (DAKO) and 1% BSA was added to eachwell and further allowed to react at room temperature for 1 hour. Theplate was sufficiently washed with a PBST solution, an then acitrate-phosphate buffer (pH 5.0) containing 0.4 mg/mlorthophenylenediamine (OPD, Sigma, P-9029) and a 0.015-0.03% hydrogenperoxide solution was added and allowed to react at room temperature forcolor development.

Then, the reaction mixture was added with a 1 N H₂SO₄ solution to stopthe reaction and measurement was performed at a measurement wavelengthof 490 nm and a reference wavelength of 650 nm. The measurement resultsare shown in FIG. 2. The reactivity of the monoclonal antibody derivedfrom the hybridoma clone AHGA-D to HGFA bonded to nafamostat mesylate,which is a protease inhibitor, was markedly lowered.

EXAMPLE 8 Preparation of Polyclonal Antibody Directed to HGFA andLabeled Version Thereof

Polyclonal antibodies directed to HGFA were prepared by subcutaneouslyadministering a mixture of 100 μg each of active 36 kDa HGFA, active 98kDa HGFA and inactive HGFA prepared in Example 1 to a rabbit 7 timeswith 2-week intervals. After production of antibodies in the serum wasconfirmed, 10 μg each of the antigen was further intravenouslyadministered and the antiserum was obtained 5 days later. Further, afterprecipitation with ammonium sulfate, anti-HGFA polyclonal antibodieswere obtained by purification using protein A column. Then, the obtainedHGFA polyclonal antibodies were labeled with biotin to preparebiotin-labeled anti-HGFA polyclonal antibodies.

EXAMPLE 9 Construction of Active HGFA Specific Measurement System

The monoclonal antibody derived from the hybridoma clone AHGA-A used inExample 4 was used as a primary antibody. This monoclonal antibody wasdissolved in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at aconcentration of 30 μg/ml, added to a 96-well plate in an amount of 100μl/well and left at 4° C. for one day (about 12 hours or longer). Theprimary antibody solution was removed from this primary antibody-coatedplate, and then 250-300 μl/well of PBS(-) containing 1% BSA was addedand left at 4° C. overnight (about 12 hours) or at 37° C. for 2 hours orlonger. The blocking solution was removed from this plate, and thenactive 36 kDa HGFA or inactive 98 kDa HGFA dissolved in 100 μl of 0.15 MNaCl, 0.1% CHAPS, 0.05% Tween 20, 0.05% Az (sodium azide), 0.1% BSA, 20mM sodium phosphate buffer (pH 7.5) was added at various concentrations(0, 0.69, 2.1, 6.2, 18.5, 55.6, 167 and 500 ng/ml) and allowed to reactat 4° C. for about 2 hours or longer.

Subsequently, the plate was sufficiently washed by using a washingsolution containing 500 mM NaCl, 0.05% Tween 20 and 20 mM Tris-HCl (pH7.5), and then 100 μl of PBS(-) containing 1 μg/ml biotin-labeledanti-HGFA polyclonal antibody prepared in Example 8 and 1% BSA was addedto each well and allowed to react in an incubator at 37° C. for 2 hours.

Then, the plate was sufficiently washed by using the above washingsolution, and then 100 μl of PBS(-) containing 1% BSA in whichHRP-conjugated streptavidin (Amersham Pharmacia Biotech, Code RPN1231)was diluted at a ratio of 1:2000 was added to each well and furtherallowed to react at room temperature for 1 hour. The plate wassufficiently washed with a washing solution, and then 100 μl/well of acitric acid-phosphate buffer containing 0.4 mg/ml orthophenylenediamine(OPD, Sigma, P-9029) and 0.015-0.03% hydrogen peroxide solution (pH 5.0)was added and allowed to react at room temperature for colordevelopment.

Subsequently, the reaction mixture was added with 100 μl/well of 1 NH₂SO₄ solution to stop the reaction and measurement was performed at ameasurement wavelength of 490 nm and a reference wavelength of 650 nm.The results are shown in FIG. 3. FIG. 3A shows the concentration of HGFAin the range of 0-500 ng/ml. In FIG. 3B, the concentration range of0-55.6 ng/ml shown in FIG. 3A is enlarged.

EXAMPLE 10 Measurement of Active HGFA Amount in Blood of Healthy Subject

Sera of 144 healthy subjects were used for the measurement utilizing theactive HGFA specific measurement system prepared in Example 9. Eachserum of healthy subject was collected, frozen, stored and thawedimmediately before use in this experiment. First, on the 96-well platecoated with the primary antibody and blocked prepared in Example 9, 50μl of 0.15 M NaCl, 0.1% CHAPS, 0.05% Tween 20, 0.05% Az, 0.1% BSA, 20 mMsodium phosphate buffer (pH 7.5) was added to each well in advance, and50 μl of a serum of healthy subject or standard 36 kDa active HGFA (0,0.69, 2.1, 6.2, 18.5, 55.6, 167 or 500 ng/ml) was added to each well andallowed to react at 4° C. for about 2 hours or longer. Then, the platewas washed by using a washing solution containing 500 mM NaCl, 0.05%Tween 20 and 20 mM Tris-HCl (pH 7.5), and then 100 μl of PBS(-)containing 1 μg/ml biotin-labeled anti-HGFA polyclonal antibody preparedin Example 8 and 1% BSA was added to each well and allowed to react inan incubator at 37° C. for 2 hours. Subsequently, the plate wassufficiently washed by using the above washing solution, and 100 μl ofPBS(-) containing 1% BSA in which HRP-conjugated streptavidin (AmershamPharmacia Biotec, Code RPN1231) was diluted at a ratio of 1:2000 wasadded to each well and further allowed to react at room temperature for1 hour.

The plate was sufficiently washed with a washing solution, and then 100μl/well of a citric acid-phosphate buffer containing 0.4 mg/mlorthophenylenediamine (OPD, Sigma, P-9029) and a 0.015-0.03% hydrogenperoxide solution (pH 5.0) was added and allowed to react at roomtemperature for color development. Then, 100 μl/well of 1 N H₂SO₄solution was added to stop the reaction and measurement was performed ata measurement wavelength of 490 nm and a reference wavelength of 650 nm.Then, a calibration curve was drawn based on the relationship betweenthe standard 36 kDa active HGFA concentration and the color developmentamount to calculate the concentration in each serum of healthy subject.The results are shown in FIG. 4. The concentration in the sera of normalsubjects was in the range of 0 to 58.5 ng/ml and the average was 19ng/ml.

EXAMPLE 11 Measurement of Active HGFA Level in Blood of Patients withVarious Human Diseases

Sera of patients with various human diseases were used for measurementperformed according to the method of Example 10 by using the active HGFAspecific measurement system prepared in Example 9. The number ofpatients for each disease was 5. Each serum was collected, frozen,stored and thawed immediately before use in the experiment. The resultsare shown in Table 1. It was found that the concentration of active HGFAwas higher in blood of patients with glomerular nephritis, pancreatitis,cancer, myocardial infarction, angina pectoris and cerebral infarction,compared with that of healthy subjects (number of specimen: 9, averagevalue: 19 ng/ml). TABLE 1 Case Patient 1 Patient 2 Patient 3 Patient 4Patient 5 Glomerular 304.2 158.3 130.0 144.8 280.7 nephritisPancreatitis 108.1 100.0 152.0 109.6 117.6 Cancer 104.1 153.9 141.6 80.9105.4 Myocardial 61.6 69.2 240.7 70.1 — infarction Angina 175.5 134.0270.7 213.8 374.3 pectoris Cerebral 64.8 96.2 554.0 — — infarction

EXAMPLE 12 Method for Collecting Blood for Measuring Active HGFA andExamination of Stability

Serum, citrated plasma, heparin plasma or EDTA plasma was collected froma normal subject or serum, and citrated plasma, heparin plasma or EDTAplasma containing argatroban at a final concentration of 40 μM wasprepared. Each was stored at 4° C. or 37° C. for 12 hours. Then, theamount of active HGFA was measured by the method described in Example10. The results are shown in FIG. 5. It was found that the increase inthe amount of active HGFA due to storage of serum or plasma was less inplasma than in serum and that this increase was favorably suppressed byaddition of argatroban.

The application is based on Japanese patent application No. 2000-370435which was filed in Japan on Dec. 5, 2000 is incorporated by referenceherein in its entirety.

1-11. (canceled)
 12. A method for measuring active HGFA, comprising thestep of measuring the active HGFA specifically by an immunologicalmethod using one or more kinds of antibodies that recognize an activehepatocyte growth factor activator (HGFA) and does not substantiallyrecognize inactive HGFA.
 13. A method for measuring active HGFA,comprising the step of measuring the active HGFA specifically by animmunological method using one or more kinds of antibodies thatrecognize active HGFA activated by limited proteolysis of inactive HGFA,which is a precursor of active HGFA, between arginine at position 407and isoleucine at a position of 408 counted from the translationinitiation amino acid of inactive HGFA, and does not substantiallyrecognize inactive HGFA.
 14. A method for measuring active HGFA,comprising the step of measuring the active HGFA specifically by animmunological method using one or more kinds of antibodies thatrecognize active HGFA and does not substantially recognize inactive HGFAand a complex of active HGFA and a protease inhibitor.
 15. The methodaccording to any one of claims 12 to 14, wherein a specimen to bemeasured for active HGFA is a biological component collected from asubject or test animal suspected of having a disease.
 16. The methodaccording to claim 15, wherein the disease is organ inflammation,glomerular nephritis, cancer, myocardial infarction, angina pectoris,cerebral infarction or thrombosis.
 17. A method for detecting a disease,comprising the step of detecting or measuring active HGFA in abiological component collected from a subject suspected of having adisease.
 18. The method according to claim 17, wherein the disease isselected from the group consisting of organ inflammation, glomerularnephritis, cancer, myocardial infarction, angina pectoris, cerebralinfarction or thrombosis.
 19. The method according to claim 17 or 18,wherein the biological component is blood or a fraction or processedproduct thereof.
 20. The method according to claim 19, wherein thebiological component is plasma.
 21. The method according to claim 20,wherein the plasma is citrated plasma.
 22. The method according to claim19, wherein argatroban is added to the biological component. 23-30.(canceled)